Transfection of the primate malaria parasite Plasmodium knowlesi using entirely heterologous constructs.

The recently developed transfection systems for Plasmodium berghei and Plasmodium falciparum offer important new tools enabling further insight into the biology of malaria parasites. These systems rely upon artificial parasite-host combinations which do not allow investigation into the complex interactions between parasites and their natural hosts. Here we report on stable transfection of Plasmodium knowlesi (a primate malaria parasite that clusters phylogenetically with P. vivax) for which both natural and artificial experimental hosts are available. Transfection of this parasite offers the opportunity to further analyze the biology of antigens not only in a natural host but also in hosts that are closely related to humans. To facilitate future development of integration-dependent transfection in P. knowlesi, completely heterologous plasmids that would reduce homologous recombination at unwanted sites in the genome were constructed. These plasmids contained the pyrimethamine-resistant form of dihydrofolate reductase-thymidylate synthase (dhfr-ts) from Toxoplasma gondii or P. berghei, under control of either (a) P. berghei or (b) P. falciparum promoters. Plasmids were electroporated into mature P. knowlesi schizonts and these cells were injected into rhesus monkeys (Macaca mulatta). After pyrimethamine treatment of these monkeys, resistant parasites were obtained that contained the plasmids. Promoter regions of both P. berghei and P. falciparum controlling dhfr-ts expression were effective in conferring pyrimethamine resistance in P. knowlesi, indicating that common signals control gene expression in phylogenetically distant Plasmodium species.

Stable transfection of malaria parasite blood stages.

Genetic manipulation of malaria parasites would revolutionize the study of this group of pathogens and have implications for vaccine and drug development. This report describes the stable, drug-selectable genetic transformation of the clinically relevant intracellular blood stages of a malaria parasite. A plasmid transfection vector carrying the gene locus that encodes a drug-resistant form of the bifunctional enzyme dihydrofolate reductase-thymidylate synthase from the rodent malaria parasite Plasmodium berghei was constructed. Derivatives of this vector were introduced into merozoites of P. berghei by electroporation, and parasites were selected for successful transformation in the rodent host on the basis of resistance to pyrimethamine. The plasmids were present in a circular, unrearranged form that replicated episomally to an observed maximum of 15 copies per cell in drug-resistant populations.

Expression of a Plasmodium gene introduced into subtelomeric regions of Plasmodium berghei chromosomes.

Targeted integration of exogenous DNA into the genome of malaria parasites will allow their phenotype to be modulated by means of gene disruption or the stable expression of foreign and mutated genes. Described here is the site-specific integration through reciprocal exchange, and subsequent expression, of a selectable marker gene into the genome of the pathogenic, bloodstage forms of the rodent malaria parasite Plasmodium berghei. Stable integration of a single copy of the marker gene (retained for more than 70 generations in the absence of drug pressure) into a nontranscribed subtelomeric repeat array of different chromosomes was observed. Expression of the gene within the subtelomeres indicated that the previously recorded absence of transcription in these regions could be due to a corresponding absence of genes rather than active silencing mechanisms.

Interspecies conservation of gene order and intron-exon structure in a genomic locus of high gene density and complexity in Plasmodium.

A 13.6 kb contig of chromosome 5 of Plasmodium berghei, a rodent malaria parasite, has been sequenced and analysed for its coding potential. Assembly and comparison of this genomic locus with the orthologous locus on chromosome 10 of the human malaria Plasmodium falciparum revealed an unexpectedly high level of conservation of the gene organisation and complexity, only partially predicted by current gene-finder algorithms. Adjacent putative genes, transcribed from complementary strands, overlap in their untranslated regions, introns and exons, resulting in a tight clustering of both regulatory and coding sequences, which is unprecedented for genome organisation of PLASMODIUM: In total, six putative genes were identified, three of which are transcribed in gametocytes, the precursor cells of gametes. At least in the case of two multiple exon genes, alternative splicing and alternative transcription initiation sites contribute to a flexible use of the dense information content of this locus. The data of the small sample presented here indicate the value of a comparative approach for Plasmodium to elucidate structure, organisation and gene content of complex genomic loci and emphasise the need to integrate biological data of all Plasmodium species into the P.falciparum genome database and associated projects such as PlasmodB to further improve their annotation.

Comparative genomics in Plasmodium: a tool for the identification of genes and functional analysis.

Comparative genomics allows inferences to be drawn about the coding potential of related genomes, and the evolutionary forces that have influenced genome organisation. Early comparisons have indicated that there is significant synteny (conserved physical association of genes) between the human parasite Plasmodium falciparum and the malaria parasites of rodents, such as Plasmodium berghei. The various Plasmodium genome initiatives have now provided the opportunity to perform comparative genomics within different species of malaria parasites in more detail, allowing the discovery of orthologues and paralogues of less well conserved genes and addressing questions of conservation, evolution and structure of multi-gene families. A remarkable level of conservation is being revealed, illustrated here by a comparison of members of one of the first conserved gene families to emerge from the sequencing initiatives, the P48/45 gene family. We have identified two additional members in this family, Pf36p and Pfs38, and shown that all members are conserved in P. falciparum and P. berghei, opening the way for functional analyses in the latter more accessible rodent malaria model. In addition, it has been shown that direct comparison of a 13.6 kb contig of a chromosome of P. berghei and the orthologous region in P. falciparum reveals an unexpected high level of conservation of gene organisation and complexity. The results of this comparison highlight the value of a comparative approach to elucidate the gene content of complex loci and improve its annotation

Developmental regulation of a Plasmodium gene involves the generation of stage-specific 5' untranslated sequences.

The B7 gene of Plasmodium berghei, highly conserved within the genus Plasmodium, encodes a nuclear protein most likely involved in chromatin assembly. In this study we describe the transcription pattern of B7 during asexual multiplication and sexual differentiation of the parasites in the blood of the vertebrate host. Two alternative transcripts have been identified: one, 1.4 kb in length is specific for asexual blood stages; the other, 1.8 kb in length is specific for sexually differentiated cells (gametocytes). The processed mRNAs are identical in their coding region and differ only in their 5' untranslated regions (5' UTRs). We show here that the differences in 5' UTRs are the result of two mechanisms: (1) the use of alternative transcription initiation sites mapped at least 1.4 kb apart, which imply the existence of separate, stage-specific promoters; (2) the splicing of a 765 bp gametocyte-specific intron at the 5' UTR of the 1.8 kb transcript.

Conserved location of genes on polymorphic chromosomes of four species of malaria parasites.

The number of chromosomes and the chromosomal location and linkage of more than 50 probes, mainly of genes, have been established in four species of Plasmodium which infect African murine rodents. We expected that the location and linkage of genes would not be conserved between these species of malaria parasites since extensive inter- and intraspecific size differences of the chromosomes existed and large scale internal rearrangements and chromosome translocations in parasites from laboratory lines had been reported. Our study showed that all four species contained 14 chromosomes, ranging in size between 0.5 and 3.5 Mb, which showed extensive size polymorphisms. The location and linkage of the genes on the polymorphic chromosomes, however, was conserved and nearly identical between these species. These results indicate that size polymorphisms of the chromosomes are more likely due to variation in non-coding (subtelomeric, repeat) sequences and show that a high plasticity of internal regions of chromosomes that may exist does not frequently affect chromosomal location and linkage of genes.

Differential expression in blood stages of the gene coding for the 21-kilodalton surface protein of ookinetes of Plasmodium berghei as detected by RNA in situ hybridisation.

The developmentally regulated transcription of the gene encoding the ookinete surface protein, Pbs21, has been investigated in the rodent malaria parasite, Plasmodium berghei, by RNA in situ hybridisation using fluorescently labelled DNA probes. We used a procedure that will allow the visualisation of cytoplasmic mRNA in the parasite and of high copy DNA repeats in the nucleus. Specific hybridisation to Pbs21 mRNA occurred in the cytoplasm of female gametocytes, zygotes and ookinetes, while asexual blood stages, male gametocytes and gametes showed no fluorescence. Analysis of the transcription of the Pbs21 gene during blood stage development in two tightly synchronised parasite clones using the same methodology revealed that transcription is restricted to sexual stages and is initiated in immature gametocytes at 19 h post invasion (hpi). At this point in development it is not yet possible to discriminate between the morphology of asexual trophozoites and immature gametocytes. At 24 hpi approximately 50% of the gametocytes transcribed the Pbs21 gene and the morphology of these gametocytes was identical and female. The distribution of the mRNA encoding Pbs21 confirmed that post-transcriptional control of expression occurred in the cytoplasm by repression of translation and not through delayed transport of the message to the cytoplasm. The transcription of the Pbs21 gene is the earliest demonstrated event in gametocytogenesis in rodent malaria species to date.

Mitomycin-C is an unreliable inhibitor for study of DNA synthesis in Plasmodium.

Cytophotometric studies on DNA synthesis during asexual and sexual development of Plasmodium berghei contradicted earlier conclusions on DNA synthesis in Plasmodium which were largely based on experiments in which mitomycin-C had been used as a DNA replication inhibitor. Therefore, the effect of mitomycin on intra erythrocytic asexual development and on microgametogenesis, fertilization and zygote/ookinete development of P. berghei was studied in vitro. All DNA-synthesizing stages (schizonts, exflagellating microgametocytes and zygotes) and also DNA synthesis itself in all such stages, are totally unaffected by mitomycin concentrations 10 times higher than that which inhibits normal development of the non-DNA-synthesizing rings and trophozoites. The results are explained by the mode of action of mitomycin.

DNA synthesis in Plasmodium berghei during asexual and sexual development.

DNA contents of individual stages of Plasmodium berghei were measured by direct microfluorometry after Feulgen-pararosaniline (SO2) staining. Sporozoites, intra-erythrocytic ringforms and trophozoites (until at least 15 h after invasion) are haploid and non-synthesizing DNA. DNA is synthesized just before and during schizogony, which takes 4-6 h. Genome duplication and segregation are alternating events throughout this process. Mature micro- and macrogametocytes have DNA contents between the haploid and diploid value; most, if not all of the DNA in excess of the haploid value is synthesized during the last 5-10 h of maturation. During gametogenesis microgametocytes within 8-10 min synthesize DNA steadily and at a very high rate to more than the octoploid value while the DNA content of macrogametocytes remains constant. Fertilization in vitro takes place within 1 h after gamete formation. Within 2 h and coinciding with the onset of meiosis the zygote then synthesizes DNA up to almost the tetraploid value, after which synthesis stops during ookinete development. All the above mentioned processes of DNA synthesis are reversibly inhibited by aphidicolin (C50 from 3-13 microM). From the rate of DNA synthesis during microgametogenesis we calculated a minimum of 1300 origins of replication in the haploid genome of P. berghei.

Generation of chromosome size polymorphism during in vivo mitotic multiplication of Plasmodium berghei involves both loss and addition of subtelomeric repeat sequences.

Extensive chromosome size polymorphism arises in Plasmodium berghei during in vivo mitotic multiplication. Size differences between homologous chromosomes involve rearrangements occurring in the subtelomeric portions while internal chromosomal regions do not contribute significantly to chromosome size polymorphism. Differences in the copy number of a 2.3-kb subtelomeric repeated unit are shown to correlate with size variations, and in at least one case to account completely for the size difference between two variants of the same chromosome.

Replication, expression and segregation of plasmid-borne DNA in genetically transformed malaria parasites.

To fully exploit the transfection technology developed for Plasmodium we investigated the features of replication, expression and segregation of an episomally maintained DNA construct during a sexual blood stage development in genetically transformed parasites of P. berghei. Using DNA in situ hybridisation techniques we were able to show that the introduced DNA construct is located in the nucleus of the parasite and is not segregating uniformly during schizogony. Replication of the construct mainly takes place between 16 and 24 h after invasion of the merozoites, coinciding with chromosomal replication. Furthermore the plasmid-borne DHFR/TS gene is constitutively transcribed throughout the asexual blood stage development. Hence the DHFR/TS promoter would appear to be a useful tool in the study of (over)expression of introduced genes and performing complementation studies in transfected parasites during the complete a sexual blood stage development of P. berghei.

Characterisation of the Cdc2-related kinase 2 gene from Plasmodium knowlesi and P. berghei.

The complete sequence of the cdc2-related kinase 2 (CRK2) gene from Plasmodium knowlesi and from P. berghei was determined. In both species, the CRK2 gene is closely linked to an elongation factor 1 alpha gene. The two CRK2 proteins are highly homologous to the P. falciparum PfPK5 protein. The CRK2 gene of both species is expressed at a low level during the asexual cell-cycle within the host erythrocytes. The P. berghei CRK2 mRNA is also present in gametocytes and in stages during development in the mosquito, suggesting a role of this protein in different parts of the life cycle. A conserved sequence located in the 5' untranslated region immediately upstream of the initiator ATG has the potential to form a stem-loop structure. Although the CRK2 protein possesses most of the domains that are conserved among cdc2-proteins, neither a recombinant P. knowlesi CRK2 protein nor a recombinant P. berghei protein was able to complement a yeast cdc28ts mutant. Furthermore and in contrast to the P. falciparum PfPK5 protein, a recombinant monomeric P. knowlesi CRK2 protein showed no kinase activity.

The effect of (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine on nuclear and organellar DNA synthesis in erythrocytic schizogony in malaria.

The very effective (ID50 = 47 nM) and selective antimalarial compound (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) abruptly arrests Plasmodium falciparum-cultured schizonts at concentrations between 1 and 10 x ID50 as soon as their DNA content reaches 8 times that of the haploid ringform stage. Even very high HPMPA concentrations do not inhibit the first 2-3 rounds of schizogonic DNA replication. Also, in the presence of HPMPA, replication of the 6-kb mitochondrial and 35-kb chloroplast-like DNA proceeds normally and in close concert with each other, both to a 16-fold amount within 5 h during the trophozoite stage. Hence the in in vitro assays HPMPApp-sensitive plasmodial DNA polymerase gamma-like enzyme (IC50 = 1 microM)--assumed to be involved in mitochondrial DNA replication--is not the target of HPMPA in vivo (living parasites), nor seems to be the DNA polymerization activities of the--in vitro also HPMPA-sensitive (IC50 = 38 microM)--DNA polymerase alpha or of any other nuclear DNA polymerase of Plasmodium. In vitro assays demonstrated that HPMPApp does not act as an alternative substrate for plasmodial polymerases, contradicting the suggestion that the observed delayed inhibition of plasmodial schizogony might be the result of DNA strand breakage caused by HPMPApp incorporation. Neither do results support the idea that the HPMPA-induced arrest of DNA replication might be due to chain termination as a result of such incorporation. We investigated whether arrest of DNA replication by HPMPA in schizonts could be explained by inhibition of the DNA synthesis rate limiting ribonucleotide reductase enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

The selectable marker human dihydrofolate reductase enables sequential genetic manipulation of the Plasmodium berghei genome.

Genetic transformation of malaria parasites has been limited by the number of selectable markers available. For the rodent malaria parasite, Plasmodium berghei, only a single selection marker has been at hand, utilising the dihydrofolate reductase-thymidylate synthase gene from either P. berghei or Toxoplasma gondii to confer resistance to the anti-malarial drug pyrimethamine. Here we report the use of the human dihydrofolate reductase (hDHFR) gene as a new selectable marker, which confers resistance to the antifolate inhibitor WR99210 upon both pyrimethamine sensitive and resistant isolates of P. berghei. Transfection with circular constructs containing the hDHFR gene resulted in the generation of highly resistant parasites containing multiple copies of episomally-maintained plasmids. These parasites showed around a 1000-fold increase in resistance to WR99210 compared to the parental parasites. We were also able to generate and select transgenic parasites harbouring only a single copy of hDHFR targeted into their genome, despite the fact that these parasites showed only a fivefold increase in resistance to WR99210 compared to the parental parasites. Importantly, and for the first time with malaria parasites, the hDHFR gene could be used in conjunction with the existing pyrimethamine selectable markers. This was demonstrated by reintroducing the circumsporozoite (CS) gene into transgenic CS-knockout mutant parasites that contained the P. berghei DHFR-TS selectable marker. The development of hDHFR as a second selectable marker will greatly expand the use of transformation technology in Plasmodium, enabling more extensive genetic manipulation and thus facilitating more comprehensive studies on the biology of the malaria parasite.

Structure and expression of a post-transcriptionally regulated malaria gene encoding a surface protein from the sexual stages of Plasmodium berghei.

The sexual stage-specific protein Pbs21 of the rodent malaria parasite Plasmodium berghei, expressed on the surface of zygotes and ookinetes, has been shown to induce an effective and long-lasting transmission blocking immunity. The gene encoding Pbs21 was cloned by screening a cDNA library prepared from enriched zygotes and ookinetes using the monoclonal antibody 13.1.15, which is capable of blocking subsequent parasite sexual development in the mosquito vector. The Pbs21 gene encoded a protein of 213 amino acids which contained a putative amino-terminal signal sequence and a putative carboxy-terminal hydrophobic membrane anchor. The amino-acid sequence was characterised by a large number of cysteine residues which were organized into 4 epidermal growth factor-like domains. The spacing of the cysteine residues was highly conserved when compared to the 25-kDa ookinete proteins of Plasmodium falciparum (Pfs25), Plasmodium reichenowi (Prs25) and Plasmodium gallinaceum (Pgs25) which were approximately 45%, 45% and 40% homologous to Pbs21 respectively. The gene is located on chromosome 5 and cross-hybridizes to a similarly defined gene unit in the other rodent malaria species Plasmodium chabaudi, Plasmodium vinckei and Plasmodium yoelii. The gene is internally disposed and not in the subtelomeric region of chromosome 5. The gene is transcribed in a stage-specific manner giving rise to an abundant 1.5-kb transcript. This mRNA is synthesised in the precursor cells to female gametes (gametocytes) however the protein is observed only after activation of the gametes, suggesting that translation of the mRNA is controlled by a post-transcriptional process. The Pbs21 gene and the P. berghei parasite system provide an excellent vehicle for the study of stage-specific transcriptional and post-transcriptional control in malaria.

Identification of the transcription initiation site of the asexually expressed rRNA genes of the malaria parasite Plasmodium berghei.

The start site of the A-type ribosomal RNA transcription units of the rodent malaria parasite, Plasmodium berghei, has been identified. The two A-type units cannot be distinguished within the transcription unit, yet exist as single copies on different chromosomes. Gene transcription initiates 820 bp upstream of the A-type small subunit (SSU) ribosomal gene and two major processing sites were mapped 610 and 611 nucleotides upstream of the SSU in the external transcribed spacer region. Surprisingly the nucleotide sequence of the DNA region containing the putative ribosomal promoter lacked repetitive DNA sequences typical of ribosomal promoters. This region was further analysed by computer using programs designed to reveal sequence-dependent structural features. Comparison of DNA curvature, duplex stability and pattern of twist angle variation revealed a striking degree of conservation between the ribosomal promoters from Plasmodium and other eukaryotes.

Malaria parasites contain two identical copies of an elongation factor 1 alpha gene.

Elongation factor 1alpha (EF-1alpha) is an abundant protein in eukaryotic cells, involved chiefly in translation of mRNA on the ribosomes, and is frequently encoded by more than one gene. Here we show the presence of two identical copies of the EF-1alpha gene in the genome of three malaria parasites, Plasmodium knowlesi, P. berghei and P. falciparum. They are organized in a head-to-head orientation and both genes are expressed in a stage specific manner at a high level, indicating that the small intergenic region contains either two strong promoters or a single bidirectional one. Both genes are expressed at the same time during erythrocytic development of the parasite. This expression pattern and the 100% similarity of the two genes excludes the possibility that the duplicated genes developed in accordance to the different types of ribosomes in Plasmodium. It is more likely that the duplication reflects a gene dosage effect. Comparison of codon usage in the Cdc2-related kinase genes (CRK2) of Plasmodium, which are expressed at a very low level, with the EF-1alpha genes indicates the existence of a codon bias for highly expressed genes, as has been shown in other organisms.

Comparison of in vivo and in vitro antimalarial activity of artemisinin, dihydroartemisinin and sodium artesunate in the Plasmodium berghei-rodent model.

The in vitro and in vivo antimalarial activity of artemisinin, artesunate and dihydroartemisinin has been compared using the Plasmodium berghei-rodent model. Drugs were added to synchronized short-term in vitro cultures of the erythrocytic stages and inhibition of parasite development was determined by measuring DNA synthesis by flow cytometry. Dihydroartemisinin was the most effective drug. IC50 values of artemisinin, artesunate and dihydroartemisinin were 1.9, 1.1 and 0.3 x 10(-8) M, respectively, when drugs were present during the complete 24 h developmental cycle. IC50 values increased significantly when drugs were added to old trophozoites, indicating that the older stages are less sensitive. To determine the in vivo antimalarial activity, mice with a parasitaemia between 1% and 3% were injected intramuscularly on 3 consecutive days with a single dose of the drugs dissolved in Miglyol 812. Again dihydroartemisinin was the most effective drug in vivo, showing a cure rate of 47% at 10 mg/kg bodyweight, while with both other drugs the recrudescence rate was 100% at the same dosage. This study showed that the P. berghei-rodent model is a useful tool for accurate comparisons of the in vivo and in vitro antimalarial activity of drugs.

The conserved genome organisation of non-falciparum malaria species: the need to know more.

The current knowledge on genomes of non-falciparum malaria species and the potential of model malaria parasites for functional analyses are reviewed and compared with those of the most pathogenic human parasite, Plasmodium falciparum. There are remarkable similarities in overall genome composition among the different species at the level of chromosome organisation and chromosome number, conserved order of individual genes, and even conserved functions of specific gene domains and regulatory control elements. With the initiative taken to sequence the genome of P. falciparum, a wealth of information is already becoming available to the scientific community. In order to exploit the biological information content of a complete genome sequence, simple storage of the bulk of sequence data will be inadequate. The requirement for functional analyses to determine the biological role of the open reading frames is commonly accepted and knowledge of the genomes of the animal model malaria species will facilitate these analyses. Detailed comparative genome information and sequencing of additional Plasmodium genomes will provide a deeper insight into the evolutionary history of the species, the biology of the parasite, and its interactions with the mammalian host and mosquito vector. Therefore, an extended and integrated approach will enhance our knowledge of malaria and will ultimately lead to a more rational approach that identifies and evaluates new targets for anti-malarial drug and vaccine development.